ISL1 Common Variant rs1017 Is Not Associated with Susceptibility to Congenital Heart Disease in a Chinese Population

Introduction

Congenital heart disease (CHD) is the leading cause of infant morbidity and mortality with an incidence of 19-75 per 1000 live births, depending on the type and severity of the defects that are included (Hoffman and Kaplan, 2002). A clear picture of how the heart forms is crucial for understanding the genesis of CHD, because dysregulation of heart development is at the root of the disease (Bruneau, 2008). Accumulative evidence has suggested that heart development is controlled by an evolutionarily conserved network of transcription factors that connect signaling pathways with genes for muscle growth, patterning, and contractility. Mutations in components of the cardiac transcription gene network cause CHD, the most common human birth defect (Olson, 2006).

Recently, ISL1, a LIM homeodomain transcription factor, has become an important molecular marker for the second heart field, which is expressed early in the cardiac progenitor population and functions near the top of a transcriptional pathway essential for heart development (Kang et al., 2009). ISL1⁺ genetic marking observation suggested that embryonic ISL1-expressing cells are multipotent cardiac progenitors that give rise to three major cardiac lineages, including cardiomyocytes, smooth muscle, and endothelial cells (Laugwitz et al., 2005; Moretti et al., 2006). Moreover, lineage tracing analysis further demonstrated that ISL1 descendants contribute extensively to the heart, including to parts of the inflow tract region, the atria, and the left ventricle, as well as the outflow tract and the right ventricle (Cai et al., 2003).

Gain- and loss-of-function experiments revealed that ISL1 is required for normal heart development by regulating cardiac gene expression and morphogenesis of the heart (Dodou et al., 2004; Lin et al., 2006; Cohen et al., 2007; Lin et al., 2007; Ma et al., 2008). Ablation of ISL1 in mice results in embryonic growth arrest at E9.5 and lethality by E10.5 (Pfaff et al., 1996; Cai et al., 2003). Additionally, ISL1 mutant mice display several common forms of cardiovascular malformations, including persistent truncus arteriosus, transposition of the great arteries, tetralogy of Fallot, ventricular septal defect, and atrial septal defect, indicating their potential role in diverse forms of human CHD (Lin et al., 2006; Bu et al., 2009). Although previous evidence proved that dysregulation of ISL1 brings about the occurrence of CHD, the mechanism about which factors alter ISL1 expression remains unclear. Interestingly, a recent study gives us a clue that the most common genetic variation, single-nucleotide polymorphisms (SNPs) in ISL1 might be functional and modify ISL1 expression, causing significant increased risk of CHD in white and black/African American populations (Stevens et al., 2010).

Thus, it will be of particular interest to identify whether a common variant in ISL1 influences susceptibility to sporadic CHD in our Chinese population.

Materials and Methods

Results

The characteristics of the CHD cases and controls included in the analysis are summarized in Table 1. There were no statistically significant differences between cases and controls in terms of age and sex. Traditionally, CHD was classified into five broad categories: cyanotic heart disease, left-sided obstruction defects, septation defects, patent ductus arteriosus, and other complex abnormalities. Of the 1003 CHD cases, 260 (25.9%) had cyanotic heart disease, 633 (63.1%) had septation defects, 49 (4.9%) had patent ductus arteriosus, 56 (5.6%) had other complex abnormalities, and only 5 (0.5%) had left-sided obstruction defects.

The genotype distributions of the SNP rs1017 in the cases and the controls are shown in Table 2. The observed genotype frequency was in agreement with that expected under the Hardy-Weinberg equilibrium in the controls. Compared with rs1017 wild-type AA, rs1017 AT was not associated with the risk of CHD (OR=0.88, 95% CI=0.71-1.08; p=0.213). A similar result was also observed for rs1017 homozygote TT (OR=0.91, 95% CI=0.48-1.75; p=0.783). Taken together, there was no appreciable difference between CHD risk and variant genotypes of ISL1 rs1017 in the recessive genetic model (OR=0.88, 95% CI=0.72-1.08; p=0.213).

To further evaluate the relevance of the rs1017 SNP, we performed stratified analyses according to subjects' age, sex, and CHD classifications, we found no overall heterogeneity of risk in different subgroups (two other CHD classification methods were also used; Fig. 1).

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FIG. 1.

Forest plot representing the dichotomized effects of ISL1 rs1017 and CHD susceptibility.

Discussion

CHDs arises from abnormal heart development during embryogenesis. The regulatory mechanisms involved in establishing the early heart and regulating its morphogenesis have been extensively studied (Bajolle et al., 2009). The heart is derived from mesoderm that responds to signals from the surrounding tissue to develop into two different heart fields (Kelly and Buckingham, 2002; Buckingham et al., 2005). The earliest population of cardiac progenitors, referred to as the first heart field, gives rise to the linear heart tube and subsequently, parts of the atria and the left ventricle. Cells of the second heart field are derived from the pharyngeal mesoderm that lies anterior and dorsal to the heart tube and, subsequently, gives rise to the outflow tract, right ventricle, part of the atria, and a few portions of the left ventricle (Mjaatvedt et al., 2001; Waldo et al., 2001; Brand, 2003).

The LIM homeodomain transcription factor ISL1 marks a cell population that constitutes the second heart field and should, thus, represent a true endogenous cardiac progenitor cell (Bu et al., 2009). In vivo cell lineage tracing in mouse embryos has confirmed that ISL1⁺ progenitors contribute to more than two-thirds of the cells in the embryonic heart (Cai et al., 2003; Moretti et al., 2006; Sun et al., 2007) and suggested that purified populations of ISL1⁺ progenitor cells are capable of self-renewal and differentiation into cardiomyocytes, smooth muscle, and endothelial lineages (Qyang et al., 2007), and throughout the inflow tract region, the atria, and the left ventricle, as well as the outflow tract and the right ventricle (Genead et al., 2010).

Functional analysis showed that ISL1 is required for proliferation, survival, and migration of cardiac progenitors into the forming heart. Inhibition of ISL1 results in growth arrest or exhibits severely defects in the outflow tract, right ventricle, atria, and inflow tract (Brade et al., 2007). More importantly, a recent publication by Stevens et al. (2010) demonstrated that two different SNPs (rs1017, rs3762977) in ISL1 contribute to risk of CHD in white or black/African American population, respectively. Specifically, within ISL1, rs1017 significantly increased the risk of CHD in a white population, while no association was observed in a black/African American population. Similarly, increased CHD risk was observed for rs3762977 in a black/African American population but not for a white population (Stevens et al., 2010).

To investigate the relationship between an ISL1 common variant and CHD risk in the Chinese population, we searched the public SNP database (the NCBI dbSNP database, build 127; www.ncbi.nlm.nih.gov/projects/SNP/) to select a common (i.e., minor allele frequency >0.05) SNP in the ISL1 gene in our Chinese population and found only one SNP rs1017 agreement to these criteria. Logistic-regression analysis showed that no association was found between rs1017 and CHD risk. To diminish the potential confounding agents, we performed stratified analyses according to subjects' age, sex, and CHD classifications, and no overall heterogeneity of risk in different subgroups was observed.

Differences in risk estimate for ISL1 polymorphism rs1017 in association with CHD might be caused by multiple factors, including etiologic heterogeneity between populations, study design, type of heart defects, and lack of information on potential effect modifiers. In the present study, the case-control design reduced the possibility of finding a chance association. Strengths of the study also include a relatively large sample size, clear exclusion criteria for cases, and matching criteria between cases and controls.

In summary, cardiac development is a complicated process, involving the expression of many genes at different times, spaces, and orders. Now that some of the genes involved in the main forms of CHD have been identified, a new challenge is to find which common polymorphisms in these genes might be causal.